In eukaryotes 5S rRNA genes are transcribed by RNA polymerase III. These genes occur in D. discoideum on the ca. 90 copies of an extrachromosomal palindrom together with the other ribosomal RNAs, which are generally transcribed by RNA polymerase I. A 5S rRNA gene has been isolated and its nucleotide sequence as well as the organization of the gene relative to the RNA polymerase I operon has been determined. The sequence of the coding region corresponds exactly to an earlier published 5S rRNA sequence. The genes are located just downstream from the 26S RNA and transcription orientations of the pol I genes and the pol III gene point into the same direction, away from the centromer of the palindrom. The isolated gene appears to be functional since it serves as a specific target for a nuclear protein, most likely TFIIIA. A genomic copy of a 5S rRNA pseudogene has been isolated from the D. discoideum strain V12. This pseudocopy contains nucleotides 52 to 82 of a bona fide 5S rRNA gene with only three mismatches. It resides 78 nucleotides downstream from the glu13(UUC) tRNA gene which in the D. discoideum strain V12 is associated with the retrotransposable element DRE.